Fig. 8. Morphological analysis of the postnatal cerebellum and determination of cell loss. Examination of the cerebellum of P4 animals by in situ RNA hybridization with a calbindin D 28k probe (A,B) showed a significant reduction in the length of the Purkinje cell layer in mutant (A) compared with control (B) animals (reduced by 50%) without obvious changes in cell density. (C-F) Morphological analysis of the cerebellum from adult homozygous Floxed Notch1 En2-Cre mutant (C,E) and control, homozygous Floxed Notch1, littermates (D,F) revealed a marked reduction in the size and foliation of the medial cerebellum (arrow, C). Histological examination of midline sagittal sections showed aberrant lobulation of the adult mutant cerebellum (E) with a loss of lobe VII compared with control (F) littermates. (G-I) In situ RNA hybridization with a calbindin D 28K probe (G,H) and quantification of lobe length (I), revealed a significant (*; P<0.05 Student’s t-test) reduction in the length of lobe 2/3 and a highly significant (**; P<0.01 Student’s t-test) reduction in the other lobes of the mutant (black bars) with the exception of lobe 10. Owing to the loss of lobe 7, lobe 6 and 7 were measured together, from the posterior end of lobe 5 to the anterior end of lobe 8. Analysis of Purkinje cell number and density (J; data not shown) in adult animals showed a highly significant (**) reduction in cell number within the anterior (A, lobes 2-5) and central (C, lobes 6-8) lobes of the mutant (black bars) as well as over the total cerebellum, but no significant difference in the posterior lobes (P, lobes 9 and 10). (K,L) Lineage tracing using the Cre-reporter transgene R26R confirmed that few Notch1-deficient, ß-galactosidase-expressing Purkinje cells were apparent in the medial cerebellum of adult mutants (homozygous Floxed Notch1 En2-Cre and R26R) (arrow, K), compared with control animals carrying heterozygous Floxed Notch1 En2-Cre and R26R transgenes (L). C, inferior colliculus; GL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Scale bars: in A, 1 mm for A,B; in E, 1 mm for E-H; in K, 1 mm for K,L.
